TRPV cation channels have been ascribed roles in nocioception and the induction of neurogenic inflammation. We hypothesized that the intense pain and constellation of neuro-inflammatory effects following cnidarian envenomation in humans are mediated by TRPV channel-sensors, and that cnidarian venoms contain novel components that are active on TRPV channels. Recently, we have demonstrated TRPV1 activity in tentacle extracts of all major classes of Cnidaria. Since TRPV cation channels have been ascribed roles in nocioception and the induction of neurogenic inflammation, we hypothesized that certain effects of cnidarian envenomation are mediated by TRPV channel-sensors and that cnidarian venoms contain novel neuroactive and immunoactive pharmacophores which are active at TRPVs. Another compelling argument in favor of a role for TRPVs in cnidarian envenomation is the marked chemical conservation between known ligands for TRPVs and components of cnidarian venoms. This revised exploratory research proposal, submitted in response to PAR-07-048 (Drug Discovery for Nervous System Disorders), seeks to identify and characterize novel pharmacophores that target TRPV cation channels in the venom of the cnidarian, Carybdea alata. Our experimental plan employs bioassay-directed fractionation methods, and combined spectroscopic approaches for the detection, purification and characterization of newfound TRPV1 pharmacophores. Specific Aim 1. Screen for TRPV-active compounds in C. alata venom. Low- to medium-throughput screening protocols, which integrate novel biochemical and conventional electrophysiological TRPV assays, will be employed to identify TRPV1 agonists in C. alata venom. Specific Aim 2. Purify and characterize TRPV pharmacophores from C. alata venom. These early metazoan TRPV pharmacophores will be isolated using paired biochemical purification/bioassay techniques and characterized by high-performance liquid chromatography (HPLC), gas chromatography (GC), and mass spectrometry (MS), and nuclear magnetic resonance (NMR) spectroscopy. Our focus on the cnidarian system minimizes the inherent risk in this type of natural product discovery effort. That is, the outcome from probing these ancient metazoans for novel structures with TRPV1 activity will not simply recapitulate previously reported bioactive compounds. The dual impact of this work will be to provide much-needed, novel pharmacology for TRPV channels, and to gain mechanistic insights into the pathological effects of cnidarian venoms. In view of recent advances which demonstrate the marked therapeutic potential of TRPV1 agonists, as well as antagonists, in the treatment of pain associated with chronic inflammation and surgery, the potential therapeutic utility of novel TRPV1 pharmacophores from cnidaria is extremely high. TRPV proteins are targets for the development of new pain medications. The venom of the Hawaiian box jelly fish contains potentially novel compounds which target TRPV channels. We will explore the chemical diversity of this venom to discover new lead compounds for the management of neurological disorders.